Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA 92093, USA.
Halıcıoğlu Data Science Institute, University of California San Diego, La Jolla, CA 92093, USA.
Chembiochem. 2023 Aug 15;24(16):e202200788. doi: 10.1002/cbic.202200788. Epub 2023 May 24.
Base editors are genome editing tools that enable site-specific base conversions through the chemical modification of nucleobases in DNA. Adenine base editors (ABEs) convert A ⋅ T to G ⋅ C base pairs in DNA by using an adenosine deaminase enzyme to modify target adenosines to inosine intermediates. Due to the lack of a naturally occurring adenosine deaminase that can modify DNA, ABEs were evolved from a tRNA-deaminating enzyme, TadA. Previous experiments with an ABE comprising a wild-type (wt) TadA showed no detectable activity on DNA, and directed evolution was therefore required to enable this enzyme to accept DNA as a substrate. Here we show that wtTadA can perform base editing in DNA in both bacterial and mammalian cells, with a strict sequence motif requirement of TAC. We leveraged this discovery to optimize a reporter assay to detect base editing levels as low as 0.01 %. Finally, we used this assay along with molecular dynamics simulations of full ABE:DNA complexes to better understand how the sequence recognition of mutant TadA variants change as they accumulate mutations to better edit DNA substrates.
碱基编辑器是一种基因组编辑工具，可通过化学修饰 DNA 中的核碱基来实现特定位置的碱基转换。腺嘌呤碱基编辑器（ABE）通过使用腺苷脱氨酶将靶腺苷修饰为肌苷中间体，将 A ⋅ T 转换为 G ⋅ C 碱基对。由于缺乏能够修饰 DNA 的天然存在的腺苷脱氨酶，ABE 是从 tRNA 脱氨酶 TadA 进化而来的。以前使用包含野生型（wt）TadA 的 ABE 的实验表明，该酶对 DNA 没有可检测的活性，因此需要定向进化才能使该酶能够接受 DNA 作为底物。在这里，我们表明 wtTadA 可以在细菌和哺乳动物细胞中在 DNA 中进行碱基编辑，严格的序列基序要求为 TAC。我们利用这一发现优化了报告基因检测，以检测低至 0.01%的碱基编辑水平。最后，我们使用该测定法以及全 ABE:DNA 复合物的分子动力学模拟，更好地了解突变型 TadA 变体的序列识别如何随着突变的积累而改变，从而更好地编辑 DNA 底物。
